Heme controls the synthesis of protein in reticulocytes. In heme-deficiency, there is diminished initiation of protein synthesis. The principal mechanism of the inhibition of initiation of protein synthesis is the phosphorylation of the alpha -subunit of the eukaryotic initiation factor 2, eIF-2 alpha. In addition to heme-deficiency, oxidized glutathione (GSSG) and low levels of double stranded RNA inhibit initiation by promoting phosphorylation of eIF-2 alpha.
The translation of mRNA in eukaryotic cells occurs in the cytoplasm. In the first step of initiation, free 80 S ribosomes are in equilibrium with their 40 S and 60 S subunits. In the presence of eIF-3, 40 S subunits bind the eIF-3 and eIF-4C to form a 43 S ribosomal complex; the binding of eIF-3 and eIF-4C to the 40 S subunit inhibits the joining of the 60 S subunit.
In the next step, eIF-2 binds GTP and the initiator tRNA, Met-tRNA f, in a ternary complex. The binding by eIF-2 is specific for both guanine nucleotides and for Met-tRNA f. The ternary complex now binds to the 43 S ribosomal complex to form the 43 S preinitiation complex. The 43 S preinitiation complex binds mRNA in an ATP-dependent reaction in which eIF-4A, eIF-4B, and eIF-4F form a complex with the mRNA. The product of the binding of mRNA to the 43 S structure is bound close to the ribosome and the AUG initiator codon is downstream from the cap structure.
The joining of the 48 S preinitiation complex and the 60 S subunit is catalyzed by eIF-5 which has a ribosome-dependent GTPase activity. The joining reaction is accompanied by the release of the initiation factors eIF-3 and eIF-4C, eIF-2 is translocated to 60 S subunit as a binary complex, elF2-GDP. The product of the joining reaction is the 80 S initiation complex. Formation of the active 80 S initiation complex is the final step in initiation. The Met-tRNA f is positioned in the P (peptidyl) site on the ribosome for the start of polypeptide elongation.
The sequence of steps in the process of initiation affords several opportunities for regulation. These include the recycling of eIF-2 after its release as the eIF-2-GDP complex; the formation of the ternary complex; and the relative affinities of mRNAs for eIF-2 and for eIF-4A, -4B, and -4F in determining the relative rates of translation of the mRNAs.
Heme-deficiency inhibited initiation of protein synthesis is characterized by a brief period of control linear synthesis, followed by an abrupt decline in this rate and by disaggregation of polyribosomes, associated with a decrease in the formation of the eIF-2-Met-tRNA f -GTP ternary complex and the 40 S-eIF-2 Met-tRNA f -GTP 43 S initiation complex. The fundamental mechanism for the inhibition is the activation of cAMP independent protein kinases that specifically phosphorylate the 38-kDa alpha-subunit of eIF-2 (elF-2 alpha). Dephosphorylation of eIF-2 alpha accompanies the recovery of protein synthesis upon addition of hemin to inhibited heme-deficient lysates.
The heme-regulated eukaryotic initiation factor 2 alpha (elF-2 alpha) kinase, also called heme-regulated inhibitor (HRI), plays a major role in this process. HRI is a cAMP-independent protein kinase that specifically phosphorylates the alpha subunit (elF-2 alpha ) of the eukaryotic initiation factor 2 (eIF-2). Phosphorylation of elF-2 alpha in reticulocyte lysates results in the binding and sequestration of reversing factor RF, also designated as guanine nucleotide exchange factor or eIF-2 B, in a RF-eIF-2 (alpha P) complex; the unavailability of RF, which is required for the exchange of GTP for GDP in the recycling of eIF-2 and in the formation of the eIF-2-Met-tRNA f -GTP ternary complex, resulting in the cessation of the initiation of protein synthesis.
Although the mechanism of regulation of protein synthesis by HRI has been extensively studied, little is known about the structure and regulation of HRI itself. Chen, J. -J., et al., Proc. Natl. Acad. Sci., USA 88:315-319 (1991) previously reported the amino acid sequences of three tryptic peptides of heme-reversible HRI. HRI peptide P-52 contains the sequence -Asp-Phe-Gly-, which is the most highly conserved short stretch in conserved domain VII of protein kinases as presented by Hanks, et al., Science 241:42-52 (1988). The N-terminal 14 amino acids of HRI peptide P-74 show 50-60% identity to the conserved domain IX of kinase-related transforming proteins. These findings are consistent with the autokinase and eIF-2 alpha kinase activities of HRI. As reported by Pal et al., Biochem. 30:2555-2562 (1991), this protein appears to be erythroid-specific and antigenically different in different species.
In view of the activity and relationships of HRI to other protein kinases involved in cellular transformation, it would be advantageous to have the nucleic acid sequence encoding HRI. However, since the gene is only expressed during a very limited time period, i.e., during erythroid differentiation, and in an extremely minuscule amount, this was not a simple process. Moreover, even though three peptides isolated by tryptic digest had been sequenced, it was not clear if these were from HRI or from a contaminant of the HRI preparation. Obtaining a library containing a full length HRI cDNA is also difficult.
Chen et al. have disclosed the nucleotide sequence for DNA encoding HRI from rabbit reticulocytes. U.S. Pat. Nos. 5,690,930 and 5,525,513. However, due to differences between species, compounds which may affect the activity of the rabbit HRI may not have the same effect on human HRI. Therefore, to use HRI in humans or to identify compounds which affect the activity of human HRI, it is essential that isolated human HRI can be produced and that the sequence of human HRI is determined.
It is therefore an object of the present invention to provide a nucleic acid sequence encoding human HRI.
The present invention provides an isolated nucleic acid sequence encoding the human heme-regulated initiation factor 2 alpha kinase.
The present invention also provides a pharmaceutical composition having the heme-regulated initiation factor 2 alpha kinase in combination with a suitable pharmaceutical carrier for administration to cells.
In a further embodiment, the invention provides a method for inhibiting protein synthesis, inducing cellular differentiation, or inhibiting infection in human cells. An effective amount of a heme-regulated initiation factor 2 alpha kinase is administered to the cells.
Yet another embodiment of the invention is a method for modulating heme-regulated initiation factor 2 alpha kinase activity, by administering an effective amount of an antibody or a receptor protein which binds to heme-regulated eukaryotic initiation factor 2 alpha kinase to cells.
Another aspect of the invention is a method for determining the level of heme-regulated initiation factor 2 alpha kinase expression, by contacting a biological sample with a nucleic acid molecule which specifically binds to a gene encoding human heme-regulated initiation factor 2 alpha kinase.